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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
24 August 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Silver docosanoate
EC Number:
219-641-4
EC Name:
Silver docosanoate
Cas Number:
2489-05-6
Molecular formula:
C22H44O2.Ag
IUPAC Name:
silver(1+) docosanoate
Constituent 2
Reference substance name:
CH03220
IUPAC Name:
CH03220
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): CH03220
- Molecular formula (if other than submission substance): C22H4402.Ag
- Molecular weight (if other than submission substance):447.44
- Physical state: white powder
- Analytical purity: 100 wt % silver docosanoate
- Lot/batch No.: CH03220/AJ
- Storage condition of test material: at room temperature, protected from light
- Stability under storage conditions: stable
- Expiry date: 25 nov 2012 (1 year after receipt of the test substance)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Standard ModelTM (EPISKIN-SM)
- Tissue batch number(s): 12-EKIN-004
- Production date: /
- Shipping date: January 24, 2012
- Delivery date: January 24, 2012
- Date of initiation of testing: January 24, 2012


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out at 37.0 ± 1.0°C (actual range 36.3 - 37.4°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not indicated
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml MTT-solution (0.3 mg/ml in PBS)
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Histology scoring: 21.3 +- 0.3 (CV = 1.2%)
IC 50 determination: 2.1 mg/ml
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination:
On blood of the same donors, we have verified:
* absence of HIV1 and 2 antibodies (Architect Abott)
* absence of hepatitis C antibodies (Architect Abott)
* absence of hepatttis B antigen HBs (Architect Abott)
on epidermal cells of the same donors, we have verified the absernce of bacteria, fungus and mycoplasma.
- Reproducibility:
Histology: probability 0.95 that 100% of the batch > 20
IC 50: probability 0.95 that IC 50 >= 2.0 mg/ml (threshold value)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-corrosive to skin if
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10.7 - 12.9 mg
- Concentration (if solution): /

VEHICLE
no vehicle

NEGATIVE CONTROL
Phosphate buffered saline (PBS, Merck KGaA, Darmstadt, Germany).
25 μl PBS

POSITIVE CONTROL
5% (aq) Sodium dodecyl sulfate (SDS, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) [CAS Number 151-21-3] in PBS.
25 μl 5% SDS
Duration of treatment / exposure:
exposure period of 15 ± 0.5 minutes at room temperature
Duration of post-treatment incubation (if applicable):
skin tissues were incubated for 42 hours at 37°C.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
99
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Because no colour change was observed it was concluded that CH03220 did not interact with MTT.
The positive control had a mean cell viability after 15 minutes exposure of 6%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: see 'any other information on materials and methods incl. tables' above
Conclusions:
it is concluded that this OECD 439 test is valid and that CH03220 is non-irritant in the in vitro skin irritation test (EpiSkin).
Executive summary:

In vitro skin irritation test with CH03220 using a human skin model.

This report describes the ability of CH03220 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)). The possible skin irritation potential of CH03220 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch CH03220/ AJ of CH03220 was a white powder with a purity of approximately 100wt% silver docosanoate. Skin tissue was moistened with 5 μl of Milli-Q water and 10.7 to 12.9 mg of CH03220 was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with CH03220 compared to the negative control tissues was 99%. Since the mean relative tissue viability for CH03220 was above 50% after 15 minutes treatment CH03220 is considered to be non-irritant.

The positive control had a mean cell viability of 6% after 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that CH03220 is non-irritant in thein vitroskin irritation test under the experimental conditions described in this report.